Copper Supplementation for Control of Glycosylation in Mammalian Cell Culture Process

ABSTRACT

The present invention pertains to a cell culture medium comprising copper as a media supplement, which was shown to control recombinant protein glycosylation and methods of using thereof. The present invention further pertains to a method of controlling or manipulating glycosylation of a recombinant protein of interest in a large scale cell culture.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention pertains to a cell culture medium comprisingcopper as a media supplement that was shown to control recombinantprotein glycosylation and methods of using thereof. The presentinvention further pertains to a method of controlling or manipulatingglycosylation of a recombinant protein of interest in a large scale cellculture, comprising controlling or manipulating the concentration ofcopper in the cell culture medium.

Background Art

Over the last few decades, much research has focused on the productionof therapeutic recombinant proteins, e.g., monoclonal antibodies. Whilemedia containing sera or hydrolysates has been utilized, chemicallydefined media were also developed in order to eliminate the problematiclot-to-lot variation of complex components (Luo and Chen, Biotechnologyand Bioengineering 97(6):1654-1659 (2007)). An improved understanding ofcell culture has permitted a shift to chemically defined medium withoutcompromising growth, viability, titer, etc. To date optimized chemicallydefined processes have been reported with titers as high as 7.5-10 g/L(Huang et al., Biotechnology Progress 26(5):1400-1410 (2010); Ma et al.,Biotechnology Progress 25(5):1353-1363 (2009); Yu et al., Biotechnologyand Bioengineering 108(5):1078-1088 (2011)). In general, the high titerchemically defined processes are fed batch processes with cultivationtimes of 11-18 days. The process intensification has been achievedwithout compromising product quality while maintaining relatively highviabilities.

Achievement of a robust, scalable production process includes more thanincreasing the product titer while maintaining high product quality. Theprocess must also predictably require the main carbohydrate sourceremain constant, such that the feeding strategy does not need to changeacross scales. As many processes use glucose as the main carbohydrate,and have lactate and ammonium as the main byproducts, the time course ofthese three critical chemicals should also scale.

A number of reports have demonstrated mammalian host cell-specificprocessing of N-glycans associated with recombinant proteins (James etal., Bio/Technology, 13:592-596 (1995); Lifely et al., Glycobiology,5:813-822 (1995)). These differences may be important for therapeuticproteins as they can directly alter the antigenicity, rate of clearancein vivo, and stability of recombinant proteins (Jenkins et al., NatureBiotechnol. 14:975-981 (1996)). Thus, it is important not only to beable to characterize glycans bound to a therapeutic recombinant proteinto predict the consequences for in vivo safety and efficacy, but also tounderstand the cellular controls underpinning glycan processing in apotential host cell enabling the implementation of appropriatestrategies to control cellular glycosylation (Grabenhosrt et al.,Glycoconjug. J., 16:81-97 (1999); James and Baker, Encyclopedia ofbioprocess technology: Fermentation, biocatalysis and bioseparation. NewYork: John Wiley & Sons. p. 1336-1349 (1999)).

Thus, there is a need in the art for identification of methods that canpredictably control glycosylation of proteins of interest.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method forachieving a predetermined galactosylation profile of an anti-α4-integrinantibody comprising adjusting the concentration of copper in a cellculture to achieve a target copper concentration range, wherein the cellculture comprises host cells producing the anti-α4-integrin antibody.

In another aspect, the invention is directed to a method for achieving apredetermined galactosylation profile of an anti-α4-integrin antibodycomprising (i) determining a copper concentration in a component of acell culture medium, (ii) if the copper concentration is below a targetcopper concentration range, supplementing the component of the cellculture medium with copper to achieve a copper concentration within thetarget copper concentration range, (iii) producing a cell culture mediumusing the component of cell culture medium with the target copperconcentration, and (iv) culturing a recombinant host cell producing ananti-α4-integrin antibody in the cell culture medium comprising the cellculture medium component.

In another aspect, the invention is directed to a method for achieving apredetermined galactosylation profile of an anti-α4-integrin antibodycomprising (i) determining a copper concentration in a component of acell culture medium, (ii) if the copper concentration is below a targetcopper concentration range, adding copper to the component of the cellculture medium to achieve a copper concentration within the targetcopper concentration range, (iii) producing a cell culture medium usingthe component of cell culture medium with the target copperconcentration, and (iv) culturing a recombinant host cell producing ananti-α4-integrin antibody in the cell culture medium comprising the cellculture medium component with the target copper concentration.

In another aspect, the invention is directed to a method for optimizinga cell culture medium for the production of an anti-α4-integrin antibodycomprising (i) determining the amount of copper in a cell culture mediumor a component used to produce a cell culture medium, and (ii) if theamount of copper is below a target range, supplementing the cell culturemedium or the component of the cell culture medium with copper toachieve an amount of copper within the target range, wherein the targetrange is sufficient to produce anti-α4-integrin antibodies with apredetermined galactosylation profile.

In another aspect, the invention is directed to a method for obtaining acomponent for a cell culture medium comprising (i) measuring the amountof copper in a hydrolysate from yeast and (ii) if the amount of copperis below a target range, supplementing the yeast hydrolysate with copperto achieve an amount of copper within the target range. In oneembodiment, the method comprises supplementing the cell culture withcopper if the copper concentration in the cell culture is below thetarget copper concentration range. In another embodiment, the copperused to supplement the cell culture, cell culture medium component, orhydrolysate from yeast is copper (II) sulfate.

In one embodiment, the predetermined galactosylation profile of theanti-α4-integrin antibodies comprises 13 to 32% galactosylation. In afurther embodiment, the predetermined galactosylation profile of theanti-α4-integrin antibody comprises 13.4 to 31.8% galactosylation. In afurther embodiment, the predetermined galactosylation profile of theanti-α4-integrin antibody comprises 15 to 31% galactosylation. In afurther embodiment, the predetermined galactosylation profile of theanti-α4-integrin antibody comprises 25% galactosylation.

In one embodiment, the target copper concentration range in the cellculture is 1 ppm to 2.4 ppm. In another embodiment, the target copperconcentration range in the cell culture is at day 0 between 20 nM and 50nM and at day 11 between 200 nM and 500 nM. In another embodiment, thetarget copper concentration is maintained through a feedback loop. Inanother embodiment, the copper concentration is constantly monitored andmaintained within the target copper concentration range. In anotherembodiment, the target copper concentration is achieved with a singledose of copper.

In one embodiment, the component of the cell culture medium is a yeasthydrolysate. In a preferred embodiment, the hydrolysate is yeastolate.

In another embodiment, the anti-α4-integrin antibodies produced byrecombinant host cells cultured in cell culture medium comprising theyeast lysate comprise a predetermined galactosylation profile. In oneembodiment, the anti-α4-integrin antibody is produced by a eukaryotichost cell. In a preferred embodiment, the eukaryotic host cell is amammalian host cell.

In one embodiment, the anti-α4-integrin antibody is produced at amanufacturing scale. In another embodiment, the copper concentrationalters the levels of the isoform variants of the anti-α4-integrinantibody. In one embodiment, the anti-α4-integrin antibody isnatalizumab.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. In the absence of copper, significant shifts in galactosylationand pI isoforms were identified among natalizumab product qualitycharacteristics. Peak 5, which represents a single C-terminal lysineresidue was specifically noted to shift significantly. Galactosylation(Gal) approached its upper specification. FIG. 1 shows the % Gal and %Peak 5 levels of various large scale manufacturing (LSM) batches.

FIG. 2. Comparison of observed versus predicted values for MAb pool peakisoforms from MFM-133-12-R060.

FIGS. 3A-3D. %Peak 5 was plotted against lactate production on Day 8(3A). % Gal was plotted against lactate production on Day 8 (3B). % Peak5 was plotted against Day 8 pH (3C). % Gal was plotted against Day 8 pH(3D).

FIGS. 4A-4B. Impact of copper concentration on Peak 5 levels versus Day8 pH (4A). Impact of copper concentration on Gal levels versus Day 8 pH.Experiments were all conducted in shake flasks and overlaid on largescale manufacturing (LSM) data. Experiment 1 includes up to 200 nM ofcopper at 5% CO₂ in the culture. Experiment 2 includes up to 150 nM ofcopper at 5% CO₂ in the culture. Experiment 3 includes between 70 and100 nM of copper at 3% CO₂ in the culture. Experiment 4 includes 70 nMof copper at 7% CO₂ in the culture.

FIGS. 5A-5D. Correlation between lactate levels and copper in TCyeastolate of large scale manufacturing (LSM) batches (5A). Correlationbetween Day 8 pH and copper in TC yeastolate of LSM batches (5B).Correlation between Peak 5 levels and copper in TC yeastolate of LSMbatches (5C). Correlation between Gal levels and copper in TC yeastolateof LSM batches (5D).

FIG. 6. Copper concentrations were measured in Facility A and B nutrientfeeds using the same TC yeastolate lot.

FIGS. 7A-7C. VCD profile of production shake flask cultures using TCyeastolate lots high/low supplemented with copper (7A). Lactate profileof production shake flask cultures using TC yeastolate lots high/lowsupplemented with copper (7B). pH profile of production shake flaskcultures using TC yeastolate lots high/low supplemented with copper(7C).

FIGS. 8A-8B. % Peak 5 was plotted against Day 8 pH of production shakeflask cultures using TC yeastolate lots (8A). % Gal was plotted againstDay 8 pH of production shake flask cultures using TC yeastolate lots(8B). Shake flask results plotted with large scale manufacturing (LSM)data.

FIGS. 9A-9C. Impact of copper levels on the viable cell density (VCD)profile of production shake flask cultures using Bio Springer yeastolatelots high/low supplemented with copper (9A). Impact of copper levels onlactate levels of production shake flask cultures using Bio Springeryeastolate lots high/low supplemented with copper (9B). Impact of copperlevels on pH of production shake flask cultures using Bio Springeryeastolate lots high/low supplemented with copper (9C).

FIGS. 10A-10B. Impact of copper levels on Peak 5 levels on Day 8 ofproduction shake flask cultures using Bio Springer yeastolate lots(10A). Impact of copper levels on Gal levels on Day 8 of productionshake flask cultures using Bio Springer yeastolate lots (10B). Shakeflask results plotted with large scale manufacturing (LSM) data.

FIGS. 11A-11B. Impact of copper levels on cell growth in productionshake flasks. The VCD results for Experiment 1 (11A). The VCD resultsfor Experiment 2 (11B).

FIG. 12. Correlation between Peak5/Peak 4 and copper concentrations inTC yeastolate.

FIG. 13. Correlation between Peak5/Gal and copper concentrations in TCyeastolate.

FIG. 14. Impact of copper in TC yeastolate on Peak 5 and Gal levels. Thehighlighted region specifies the target copper range.

FIGS. 15A-15B. Impact of copper levels in yeastolate on % Aggregate (5A)and %Half Antibody (5B).

FIGS. 16A-16J. Impact of copper levels in yeastolate on IEC peaks 1-11respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the recognition that cell culturemedia supplemented with copper provides the ability to control andmanipulate the glycolsylation patterns of recombinant glycoproteinsproduced in eukaryotic cell cultures. Such glyclosylation patternsinclude, without limitation, the level of galactosylation, andvariations in isoform levels.

The present invention is also applicable to modifying the glycosylationof a recombinant glycoprotein of interest such that it falls within thequality attribute ranges for the desired product. For example, thepresent invention is applicable to modifying the glycosylation profileof a recombinant glycoprotein of interest to more closely resemble,match, or substantially match the glycosylation pattern of a referencesample of the same glycoprotein. Differences between variousmanufacturing processes can result in glycoproteins with identical aminoacid sequences having different glycosylation patterns depending on, forexample, conditions for growth, cell line used to express theglycoprotein, etc.

Provided herein are methods for achieving a predetermined glycosylationprofile of a recombinant glycoprotein of interest comprising adjustingthe concentration of copper in a cell culture to achieve a targetconcentration range, wherein the cell culture comprises host cellsproducing the recombinant glycoprotein of interest. Also provided hereinare methods for optimizing a cell culture medium for the production of arecombinant glycoprotein of interest comprising (i) determining theamount of copper in a cell culture medium or a component used to producea cell culture medium, and (ii) adjusting the concentration of copper inthe cell culture medium to achieve an amount of copper within the targetrange, wherein the target range is sufficient to produce the recombinantglycoprotein of interest with a predetermined galactosylation profile.Finally, provided herein are methods for obtaining a component for acell culture medium comprising (i) measuring the amount of copper in ahydrolysate from yeast and (ii) if the amount of copper is below atarget range, supplementing the yeast hydrolysate with copper to achievean amount of copper within the target range.

I. Definitions

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. The terms “a” (or “an”), as well as theterms “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever embodiments are described with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various embodimentsof the disclosure, which can be had by reference to the specification asa whole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

The terms “polypeptide” or “protein” as used herein refers a sequentialchain of amino acids linked together via peptide bonds. The term is usedto refer to an amino acid chain of any length, but one of ordinary skillin the art will understand that the term is not limited to lengthychains and can refer to a minimal chain comprising two amino acidslinked together via a peptide bond. If a single polypeptide is thediscrete functioning unit and does require permanent physicalassociation with other polypeptides in order to form the discretefunctioning unit, the terms “polypeptide” and “protein” as used hereinare used interchangeably. If discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” as used herein refers to the multiple polypeptidesthat are physically coupled and function together as the discrete unit.

The term “glycoprotein” refers to a polypeptide or protein coupled to atleast one carbohydrate moiety, e.g., a polysaccharide or anoligosaccharide, that is attached to the protein via anoxygen-containing or a nitrogen-containing side chain of an amino acidresidue, e.g., a serine or threonine residue (“O-linked”) or anasparagine residue (“N-linked”). The term “glycan” refers to apolysaccharide or an oligosaccharide, e.g., a polymer comprised ofmonosaccharides. Glycans can be homo- or heteropolymers ofmonosaccharide residues, and can be linear or branched.

As used herein, the “glycosylation pattern” of a recombinantglycoprotein of interest refers to various physical characteristics ofthe glycoprotein's polysaccharides or oligosaccharides, such as, e.g.,the quantity and quality of various monosaccharides present, the degreeof branching, and/or the attachment (e.g., N-linked or O-linked). The“glycosylation pattern” of a glycoprotein can also refer to thefunctional characteristics imparted by the glycoprotein'soligosaccharides and polysaccharides. For example, the extent to whichthe glycoprotein can bind to FcγRIIIa and induce antibody-dependentcellular cytotoxicity (ADCC).

“Fucosylation” refers to the degree and distribution of fucose residueson polysaccharides and oligosaccharides, for example, N-glycans,O-glycans and glycolipids. Therapeutic glycoproteins, e.g., antibodiesor Fc fusion proteins, with non-fucosylated, or “afucosylated” N-glycansexhibit dramatically enhanced antibody-dependent cellular cytotoxicity(ADCC) due to the enhancement of FcγRIIIa binding capacity without anydetectable change in complement-dependent cytotoxicity (CDC) or antigenbinding capability. In certain situations, e.g., cancer treatment,non-fucosylated or “afucosylated” antibodies are desirable because theycan achieve therapeutic efficacy at low doses, while inducing highcellular cytotoxicity against tumor cells, and triggering high effectorfunction in NK cells via enhanced interaction with FcγRIIIa. In othersituations, e.g., treatment of inflammatory or autoimmune diseases,enhanced ADCC and FcγRIIIa binding is not desirable, and accordinglytherapeutic glycoproteins with higher levels of fucose residues in theirN-glycans can be preferable. As used herein, the term “% afucose” refersto the percentage of non-fucosylated N-glycans present on a recombinantglycoprotein of interest. A higher % afucose denotes a higher number ofnon-fucosylated N-glycans, and a lower % afucose denotes a higher numberof fucosylated N-glycans.

“Sialylation” refers to the type and distribution of sialic acidresidues on polysaccharides and oligosaccharides, for example,N-glycans, O-glycans and glycolipids. Sialic acids are most often foundat the terminal position of glycans. Sialylation can significantlyinfluence the safety and efficacy profiles of these proteins. Inparticular, the in vivo half-life of some biopharmaceuticals correlateswith the degree of oligosaccharide sialylation. Furthermore, thesialylation pattern can be a very useful measure of product consistencyduring manufacturing.

The two main types of sialyl residues found in biopharmaceuticalsproduced in mammalian expression systems are N-acetyl-neuraminic acid(NANA) and N-glycolylneuraminic acid (NGNA). These usually occur asterminal structures attached to galactose (Gal) residues at thenon-reducing terminii of both N- and O-linked glycans.

“Galactosylation” refers to the type and distribution of galactoseresidues on polysaccharides and oligosaccharides. Galactose refers to agroup of monosaccharides which include open chain and cyclic forms. Animportant disaccharide form of galactose isgalactose-alpha-1,3-galactose (α-gal).

The term “undesirable side effects” refers to certain aspects andresults of glycosylation which, under certain circumstances, are to beminimized or avoided. In certain aspects, a side effect to be reduced oravoided is a substantial increase in the level of α-gal. In anotheraspect a side effect to be reduced or avoided is a substantial reductionin sialic acid levels. In various aspects the methods described hereinachieve certain glycosylation patterns without substantially affectingculture density, cell viability level, or both. In certain aspects, a“side effect” which might be undesirable in one glycoprotein, e.g., adecrease in fucose levels (increases ADCC and FcγRIIIa binding) in anantibody used to treat an inflammatory disease, might be desirable inanother glycoprotein, e.g., in an antibody used to treat cancer.

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies generated from atleast two intact antibodies, monovalent or monospecific antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody and refers to the antigenic determining variable regionsof an intact antibody. Examples of antibody fragments include, but arenot limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,single chain antibodies, and multispecific antibodies formed fromantibody fragments.

“Recombinantly expressed glycoprotein” and “recombinant glycoprotein” asused herein refer to a glycoprotein expressed from a host cell that hasbeen genetically engineered to express that glycoprotein. Therecombinantly expressed glycoprotein can be identical or similar toglycoproteins that are normally expressed in the mammalian host cell.The recombinantly expressed glycoprotein can also be foreign to the hostcell, i.e. heterologous to peptides normally expressed in the mammalianhost cell. Alternatively, the recombinantly expressed glycoprotein canbe chimeric in that portions of the glycoprotein contain amino acidsequences that are identical or similar to glycoproteins normallyexpressed in the mammalian host cell, while other portions are foreignto the host cell. In certain embodiments, the recombinant glycoproteincomprises an antibody or fragments thereof. As used herein, the terms“recombinantly expressed glycoprotein” and “recombinant glycoprotein”also encompasses an antibody produced by a hybridoma.

The term “expression” or “expresses” are used herein to refer totranscription and translation occurring within a host cell. The level ofexpression of a product gene in a host cell can be determined on thebasis of either the amount of corresponding mRNA that is present in thecell or the amount of the protein encoded by the product gene that isproduced by the cell. For example, mRNA transcribed from a product geneis desirably quantitated by northern hybridization, Sambrook et al.,Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring HarborLaboratory Press, 1989). Protein encoded by a product gene can bequantitated either by assaying for the biological activity of theprotein or by employing assays that are independent of such activity,such as western blotting or radioimmunoassay using antibodies that arecapable of reacting with the protein, Sambrook et al., MolecularCloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring HarborLaboratory Press, 1989).

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or5,821,337 can be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of a moleculeof interest may be assessed in vivo, e.g., in an animal model such asthat disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Clq) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased Clq binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “basal media formulation” or “basal media” as used hereinrefers to any cell culture media used to culture cells that has not beenmodified either by supplementation, or by selective removal of a certaincomponent.

As used herein, the terms “additive” or “supplement” refer to anysupplementation made to a basal medium to achieve the goals described inthis disclosure. An “additive” or “supplement” can include a singlesubstance, e.g., copper II sulfate, or can include multiple substances,e.g., various copper salts. The terms “additive” or “supplement” referto the all of the components added, even though they need not be addedat the same time, and they need not be added in the same way. Forexample, one or more components of an “additive” or “supplement” can beadded as a single bolus or two or more boli from a stock solution, whileother components of the same “additive” or “supplement” can be added aspart of a feed medium. In addition, any one or more components of an“additive” or “supplement” can be present in the basal medium from thebeginning of the cell culture.

The terms “culture”, “cell culture” and “eukaryotic cell culture” asused herein refer to a eukaryotic cell population, eithersurface-attached or in suspension that is maintained or grown in amedium (see definition of “medium” below) under conditions suitable tosurvival and/or growth of the cell population. As will be clear to thoseof ordinary skill in the art, these terms as used herein can refer tothe combination comprising the mammalian cell population and the mediumin which the population is suspended.

The terms “media”, “medium”, “cell culture medium”, “culture medium”,“tissue culture medium”, “tissue culture media”, and “growth medium” asused herein refer to a solution containing nutrients, which nourishgrowing cultured eukaryotic cells. Typically, these solutions provideessential and non-essential amino acids, vitamins, energy sources,lipids, and trace elements required by the cell for minimal growthand/or survival. The solution can also contain components that enhancegrowth and/or survival above the minimal rate, including hormones andgrowth factors. The solution is formulated to a pH and saltconcentration optimal for cell survival and proliferation. The mediumcan also be a “defined medium” or “chemically defined medium”—aserum-free medium that contains no proteins, hydrolysates or componentsof unknown composition. Defined media are free of animal-derivedcomponents and all components have a known chemical structure. One ofskill in the art understands a defined medium can comprise recombinantglycoproteins or proteins, for example, but not limited to, hormones,cytokines, interleukins and other signaling molecules.

The cell culture medium is generally “serum free” when the medium isessentially free of serum, or fractions thereof, from any mammaliansource (e.g. fetal bovine serum (FBS)). By “essentially free” is meantthat the cell culture medium comprises between about 0-5% serum,preferably between about 0-1% serum, and most preferably between about0-0.1% serum. Advantageously, serum-free “defined” medium can be used,wherein the identity and concentration of each of the components in themedium is known (i.e., an undefined component such as bovine pituitaryextract (BPE) is not present in the culture medium).

The term “cell viability” as used herein refers to the ability of cellsin culture to survive under a given set of culture conditions orexperimental variations. The term as used herein also refers to thatportion of cells which are alive at a particular time in relation to thetotal number of cells, living and dead, in the culture at that time.

The term “cell density” as used herein refers to that number of cellspresent in a given volume of medium.

The term “batch culture” as used herein refers to a method of culturingcells in which all the components that will ultimately be used inculturing the cells, including the medium (see definition of “medium”below) as well as the cells themselves, are provided at the beginning ofthe culturing process. A batch culture is typically stopped at somepoint and the cells and/or components in the medium are harvested andoptionally purified.

The term “fed-batch culture” as used herein refers to a method ofculturing cells in which additional components are provided to theculture at some time subsequent to the beginning of the culture process.A fed-batch culture can be started using a basal medium. The culturemedium with which additional components are provided to the culture atsome time subsequent to the beginning of the culture process is a feedmedium. A fed-batch culture is typically stopped at some point and thecells and/or components in the medium are harvested and optionallypurified.

The term “perfusion culture” as used herein refers to a method ofculturing cells in which additional components are provided continuouslyor semi-continuously to the culture subsequent to the beginning of theculture process. The provided components typically comprise nutritionalsupplements for the cells which have been depleted during the culturingprocess. A portion of the cells and/or components in the medium aretypically harvested on a continuous or semi-continuous basis and areoptionally purified.

The term “bioreactor” as used herein refers to any vessel used for thegrowth of a mammalian cell culture. The bioreactor can be of any size solong as it is useful for the culturing of mammalian cells. Typically,the bioreactor will be at least 1 liter and can be 10, 50, 100, 250,500, 1000, 2000, 2500, 3000, 5000, 8000, 10,000, 12,0000, 15,000,20,000, 30,000 liters or more, or any volume in between. For example, abioreactor will be 10 to 5,000 liters, 10 to 10,000 liters, 10 to 15,000liters, 10 to 20,000 liters, 10 to 30,000 liters, 50 to 5,000 liters, 50to 10,000 liters, 50 to 15,000 liters, 50 to 20,000 liters, 50 to 30,000liters, 1,000 to 5,000 liters, or 1,000 to 3,000 liters. A bioreactorcan be a stirred-tank bioreactor or a shake flask. The internalconditions of the bioreactor, for example, but not limited to pH andtemperature, are typically controlled during the culturing period. Thebioreactor can be composed of any material that is suitable for holdingmammalian cell cultures suspended in media under the culture conditionsof the present invention, including glass, plastic or metal. The term“production bioreactor” as used herein refers to the final bioreactorused in the production of the glycoprotein or protein of interest. Thevolume of the large-scale cell culture production bioreactor istypically at least 500 liters and can be 1000, 2000, 2500, 5000, 8000,10,000, 12,0000, 15,000 liters or more, or any volume in between. Forexample, the large scale cell culture reactor will be between about 500liters and about 20,000 liters, about 500 liters and about 10,000liters, about 500 liters and about 5,000 liters, about 1,000 liters andabout 30,000 liters, about 2,000 liters and about 30,000 liters, about3,000 liters and about 30,000 liters, about 5,000 liters and about30,000 liters, or about 10,000 liters and about 30,000 liters, or alarge scale cell culture reactor will be at least about 500 liters, atleast about 1,000 liters, at least about 2,000 liters, at least about3,000 liters, at least about 5,000 liters, at least about 10,000 liters,at least about 15,000 liters, or at least about 20,000 liters. One ofordinary skill in the art will be aware of and will be able to choosesuitable bioreactors for use in practicing the present invention.

The term “stirred-tank bioreactor” as used herein refers to any vesselused for the growth of a mammalian cell culture that has an impeller.

The term “shake flask” as used herein refers to any vessel used for thegrowth of a mammalian cell culture that does not have an impeller.

The term “hybridoma” as used herein refers to a cell created by fusionof an immortalized cell derived from an immunologic source and anantibody-producing cell. The resulting hybridoma is an immortalized cellthat produces antibodies. The individual cells used to create thehybridoma can be from any mammalian source, including, but not limitedto, rat, pig, rabbit, sheep, pig, goat, and human. The term alsoencompasses trioma cell lines, which result when progeny of heterohybridmyeloma fusions, which are the product of a fusion between human cellsand a murine myeloma cell line, are subsequently fused with a plasmacell. Furthermore, the term is meant to include any immortalized hybridcell line that produces antibodies such as, for example, quadromas (See,e.g., Milstein et al., Nature, 537:3053 (1983)).

The term “osmolality” is a measure of the osmotic pressure of dissolvedsolute particles in an aqueous solution. The solute particles includeboth ions and non-ionized molecules. Osmolality is expressed as theconcentration of osmotically active particles (i.e., osmoles) dissolvedin 1 kg of water (1 mOsm/kg H₂O at 38° C. is equivalent to an osmoticpressure of 19 mm Hg). “Osmolarity” refers to the number of soluteparticles dissolved in 1 liter of solution. Solutes which can be addedto the culture medium so as to increase the osmolality thereof includeproteins, peptides, amino acids, non-metabolized polymers, vitamins,ions, salts, sugars, metabolites, organic acids, lipids, etc. In thepreferred embodiment, the concentration of amino acids and NaCl in theculture medium is increased in order to achieve the desired osmolalityranges set forth herein. When used herein, the abbreviation “mOsm” means“milliosmoles/kg H₂O”.

The term “titer” as used herein refers to the total amount ofrecombinantly expressed glycoprotein or protein produced by a cellculture divided by a given amount of medium volume. Titer is typicallyexpressed in units of milligrams of glycoprotein or protein permilliliter of medium or in units of grams of glycoprotein or protein perliter of medium.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., cellular viability). Thedifference between said two values is, for example, less than about 50%,less than about 40%, less than about 30%, less than about 20%, and/orless than about 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein with regard to amounts or numerical values (and not asreference to the chemical process of reduction), denotes a sufficientlyhigh degree of difference between two numeric values (generally oneassociated with a molecule and the other associated with areference/comparator molecule) such that one of skill in the art wouldconsider the difference between the two values to be of statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., cellular viability). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

II. Supplementation of Cell Culture Medium to Control GlycosylationPatterns

Provided herein are methods to culture eukaryotic cells engineered toexpress a recombinant glycoprotein of interest. Specifically thisdisclosure provides methods for controlling the glycosylation patternsof a recombinant glycoprotein of interest by supplementing a tissueculture medium in which the cells are growing and/or producing therecombinant glycoprotein of interest with an additive, or culturingeukaryotic cells engineered to express a glycoprotein of interest in atissue culture medium, which has been supplemented with such anadditive. In certain embodiments, glycoproteins produced by the methodsprovided are recovered. The methods are based on the recognition thatgrowth of cells expressing a recombinant glycoprotein of interest incell culture medium supplemented with copper can result in alterationsto eukaryotic cell glycosylation patterns, such as the level ofgalactosylation. In certain embodiments, the copper added is copper (II)sulfate. In certain embodiments, the alteration of the glycosylationpattern of the recombinant glycoprotein of interest comprises a reducedlevel of galactosylation. In one embodiment, the recombinantglycoprotein of interest comprises a predetermined galactosylationprofile. In another embodiment, the recombinant glycoprotein of interestcomprising a predetermined galactosylation profile is ananti-α4-integrin antibody. In another embodiment, the predeterminedgalactosylation profile of the anti-α4-integrin antibody comprises 10 to35% galactosylation, 10 to 30% galactosylation, 10 to 20%galactosylation, 10 to 15% galactosylation, 15 to 35% galactosylation,20 to 35% galactosylation, 25 to 35% galactosylation, or 30 to 35%galactosylation. In another embodiment, the predeterminedgalactosylation profile of the anti-α4-integrin antibody comprises 13 to32% galactosylation. In another embodiment, the predeterminedgalactosylation profile of the anti-α4-integrin antibody comprises 13.4to 31.8% galactosylation. In another embodiment, the predeterminedgalactosylation profile of the anti-α4-integrin antibody comprises 15 to31% galactosylation. In another embodiment, the predeterminedgalactosylation profile of the anti-α4-integrin antibody comprises 20 to28% or 22 to 25% galactosylation. In another embodiment, thepredetermined galactosylation profile of the anti-α4-integrin antibodycomprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35% galactosylation. Inanother embodiment, the predetermined galactosylation profile of theanti-α4-integrin antibody comprises 25% galactosylation.

In one embodiment, the copper concentration in the cell culture altersthe levels of the isoform variants of the anti-α4-integrin antibody. Inone embodiment, the copper concentration in the cell culture alters thelevel of the Peak 4 isoform of the anti-α4-integrin antibody. In oneembodiment, the copper concentration in the cell culture alters thelevel of the Peak 5 isoform of the anti-α4-integrin antibody. In oneembodiment, the copper concentration in the cell culture alters thelevel of the Peak 6 isoform of the anti-α4-integrin antibody. In oneembodiment, the copper concentration in the cell culture In oneembodiment, the copper concentration in the cell culture alters thelevel of the Peak 8 isoform of the anti-α4-integrin antibody. In oneembodiment, the copper concentration in the cell culture alters thelevel of the Peak 9 isoform of the anti-α4-integrin antibody. In oneembodiment, the copper concentration in the cell culture alters thelevel of the Peak 10 isoform of the anti-α4-integrin antibody.

The present invention is applicable to altering, manipulating, orcontrolling the glycosylation pattern of a recombinant glycoprotein ofinterest to match, substantially match, approach, or more closelyresemble the glycosylation pattern of the same glycoprotein, butproduced in a different cell culture system. Recombinant glycoproteinsof interest can be produced according to the invention using variousdifferent cell culture systems, e.g., a batch culture, fed-batch culturea perfusion culture, a shake flask, and/or a bioreactor. In oneembodiment, cells expressing a recombinant glycoprotein of interest arecultured in basal medium to which the additive is introduced as a bolus,or two or more boli, from a stock solution. In another embodiment, theadditive is introduced as a component of a feed medium. In certainembodiments the cell culture comprises a growth phase and a proteinproduction phase, and the additive is introduced into the culture mediumbefore, or at the same time as, or at some point after the initiation ofthe protein production phase.

In one embodiment, a medium described herein is a serum-free medium,animal protein-free medium or a chemically-defined medium. In a specificembodiment, a medium described herein is a chemically-defined medium.

In certain embodiments, the method comprises adding CuSO₄. CuSO₄ can beadded to the culture medium in one bolus or two or more boli from astock solution to, or be added as a component of a feed medium achieve aCuSO₄ concentration in the culture medium of between about 0.01 mM andabout 1 mM CuSO₄. In certain embodiments the additive comprises CuSO₄,which can be added to the culture medium in one bolus or two or moreboli from a stock solution, or be added as a component of a feed mediumto achieve a CuSO₄ concentration in the culture medium between about0.01 mM and about 1 mM, about 0.01 mM and about 0.5 mM, about 0.01 mMand about 0.25 mM, about 0.01 mM and about 0.2 mM, about 0.01 mM andabout 0.1 mM, about 0.01 mM and about 0.05 mM, about 0.02 mM and about 1mM, about 0.05 mM and about 1 mM, about 0.1 mM and about 1 mM, about 0.2mM and about 1 mM, about 0.5 mM and about 1 mM, about 0.2 mM and about0.5 mM, or about 0.02 mM and about 0.05 mM. In another embodiment, thetarget copper concentration in the culture medium is between 0.02 mM and0.05 mM at Day 0. In another embodiment, the target copper concentrationin the culture mediums is between 0.2 mM and 0.5 mM at Day 11. Inanother embodiment, the copper concentration is constantly monitored andmaintained within the target copper concentration range. In anotherembodiment, the target concentration is maintained through a feedbackloop.

In another embodiment, the concentration of copper added to the cellculture is about 1.0 ppm to 2.5 ppm, about 1.1 ppm to 2.5 ppm, about 1.2ppm to 2.5 ppm, about 1.3 ppm to 2.5 ppm, about 1.4 ppm to 25 ppm, about1.5 ppm to 2.5 ppm, about 1.6 ppm to 2.5 ppm, about 1. 7 ppm to 2.5 ppm,about 1.8 ppm to 2.5 ppm, about 1.9 ppm to 2.5 ppm, about 2.0 ppm to 2.5ppm, about 2.2 ppm to 2.5 ppm, about 2.3 ppm to 2.5 ppm, about 2.4 ppmto 2.5 ppm, about 1.0 ppm to 2.4 ppm, about 1.0 ppm to 2.3 ppm, about1.0 ppm to 2.2 ppm, about 1.0 ppm to 2.1 ppm, about 1.0 ppm to 2.0 ppm,about 1.0 ppm to 1.9 ppm, about 1.0 ppm to 1.8 ppm, about 1.0 ppm to 1.7ppm, about 1.0 ppm to 1.6 ppm, about 1.0 ppm to 1.5 ppm, about 1.0 ppmto 1.4 ppm, about 1.0 ppm to 1.3 ppm, about 1.0 ppm to 1.2 ppm, or about1.0 ppm to 1.1 ppm. In another embodiment, the concentration of copperadded to the cell culture is 1.0 ppm to 2.4 ppm.

III. Cell Culture Compositions

The present invention further provides a cell culture compositioncomprising a medium described herein and cells, produced by the methodsprovided herein.

In one embodiment, a cell culture composition produced by the providedmethods can be a batch culture, fed-batch culture or a perfusionculture. In a specific embodiment, a cell culture composition of theinvention is a fed batch culture.

In one embodiment, a cell culture composition produced by the providedmethods comprises eukaryotic cells. In another embodiment, a cellculture composition produced by the provided methods comprises mammaliancells selected from the group consisting of CHO cells, HEK cells, NSOcells, PER.C6 cells, 293 cells, HeLa cells, and MDCK cells. In aspecific embodiment, a cell culture composition described hereincomprises CHO cells. In another specific embodiment, a cell culturecomposition described herein comprises HEK cells. In another specificembodiment, a cell culture composition described herein compriseshybridoma cells.

A cell culture composition produced by the provided methods can comprisecells that have been adapted to grow in serum free medium, animalprotein free medium or chemically defined medium. Or it can comprisecells that have been genetically modified to increase their life-span inculture. In one embodiment, the cells have been modified to express ananti-α4-integrin antibody. In a further embodiment, the cells have beenmodified to express natalizumab.

The present invention provides a method of culturing cells, comprisingcontacting the cells with a medium disclosed herein, supplementing themedium as described above, or culturing cells in a medium supplementedas described above.

Cell cultures can be cultured in a batch culture, fed batch culture or aperfusion culture. In one embodiment, a cell culture according to amethod of the present invention is a batch culture. In anotherembodiment, a cell culture according to a method of the presentinvention is a fed batch culture. In a further embodiment, a cellculture according to a method of the present invention is a perfusionculture. In certain embodiments the cell culture is maintained in ashake flask, in certain embodiments the cell culture is maintained in abioreactor.

In one embodiment, a cell culture according to a method of the presentinvention is a serum-free culture. In another embodiment, a cell cultureaccording to a method of the present invention is a chemically definedculture. In a further embodiment, a cell culture according to a methodof the present invention is an animal protein free culture.

In one embodiment, a cell culture produced by the provided methods iscontacted with a medium described herein during the growth phase of theculture. In another embodiment, a cell culture is contacted with amedium described herein during the production phase of the culture.

In one embodiment, a cell culture produced by the provided methods iscontacted with a feed medium described herein during the productionphase of the culture. In one embodiment, the culture is supplementedwith the feed medium between about 1 and about 25 times during thesecond time period. In another embodiment, a culture is supplementedwith the feed medium between about 1 and about 20 times, between about 1and about 15 times, or between about 1 and about 10 times during thefirst time period. In a further embodiment, a culture is supplementedwith the feed medium at least once, at least twice, at least threetimes, at least four times, at least five times, at least 6 times, atleast 7 times, at least 8 times, at least 9 times, at least 10 times, atleast 11 times, at least 12 times, at least 13 times, at least 14 times,at least 15 times, at least 20 times, at least 25 times. In a specificembodiment, the culture is a fed batch culture. In another specificembodiment, the culture is a perfusion culture.

A culture produced by the provided methods can be contacted with a feedmedium described herein at regular intervals. In one embodiment, theregular interval is about once a day, about once every two days, aboutonce every three days, about once every 4 days, or about once every 5days. In a specific embodiment, the culture is a fed batch culture. Inanother specific embodiment, the culture is a perfusion culture.

A culture produced by the provided methods can be contacted with a feedmedium described herein on an as needed basis based on the metabolicstatus of the culture. In one embodiment, a metabolic marker of a fedbatch culture is measured prior to supplementing the culture with a feedmedium described herein. In one embodiment, the metabolic marker isselected from the group consisting of: glucose concentration, lactateconcentration, ammonium concentration, alanine concentration, glutamineconcentration, glutamate concentration, cell specific lactate productionrate to the cell specific glucose uptake rate ratio (LPR/GUR ratio), andRhodamine 123 specific cell fluorescence. In one embodiment, an LPR/GURvalue of >0.1 indicates the need to supplement the culture with a feedmedium described herein. In a further specific embodiment, a lactateconcentration of >3 g/L indicates the need to supplement the culturewith a feed medium described herein. In another embodiment, a cultureaccording to the present invention is supplemented with a feed mediumdescribed herein when the LPR/GUR value of the culture is >0.1 or whenthe lactate concentration of the culture is >3 g/L. In a specificembodiment, the culture is a fed batch culture. In another specificembodiment, the culture is a perfusion culture.

In one embodiment, a medium described herein is a feed medium for a fedbatch cell culture. A skilled artisan understands that a fed batch cellculture can be contacted with a feed medium more than once. In oneembodiment, a fed batch cell culture is contacted with a mediumdescribed herein only once. In another embodiment, a fed batch cellculture is contacted with a medium described herein more than once, forexample, at least twice, at least three times, at least four times, atleast five times, at least six times, at least seven times, or at leastten times.

In accordance with the present invention, the total volume of feedmedium added to a cell culture should optimally be kept to a minimalamount. For example, the total volume of the feed medium added to thecell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45 or 50% of the volume of the cell culture prior to adding the feedmedium.

Cell cultures produced by the provided methods can be grown to achieve aparticular cell density, depending on the needs of the practitioner andthe requirement of the cells themselves, prior to being contacted with amedium described herein. In one embodiment, the cell culture iscontacted with a medium described herein at a viable cell density of 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95 or 99 percent of maximal viable cell density. In a specificembodiment, the medium is a feed medium.

Cell cultures produced by the provided methods can be allowed to growfor a defined period of time before they are contacted with a mediumdescribed herein. In one embodiment, the cell culture is contacted witha medium described herein at day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 of the cell culture. In another embodiment, the cell culture iscontacted with a medium described herein at week 1, 2, 3, 4, 5, 6, 7, or8 of the cell culture. In a specific embodiment, the medium is a feedmedium.

Cell cultures produced by the provided methods can be cultured in theproduction phase for a defined period of time. In one embodiment, thecell culture is contacted with a feed medium described herein at day 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the production phase.

A culture produced by the provided methods can be maintained inproduction phase for between about 1 day and about 30 days. In oneembodiment, a culture is maintained in production phase for betweenabout 1 day and about 30 days , between about 1 day and about 25 days ,between about 1 day and about 20 days, about 1 day and about 15 days,about 1 day and about 14 days, about 1 day and about 13 days, about 1day and about 12 days, about 1 day and about 11 days, about 1 day andabout 10 days, about 1 day and about 9 days, about 1 day and about 8days, about 1 day and about 7 days, about 1 day and about 6 days, about1 day and about 5 days, about 1 day and about 4 days, about 1 day andabout 3 days, about 2 days and about 25 days, about 3 days and about 25days, about 4 days and about 25 days, about 5 days and about 25 days,about 6 days and about 25 days, about 7 days and about 25 days, about 8days and about 25 days, about 9 days and about 25 days, about 10 daysand about 25 days, about 15 days and about 25 days, about 20 days andabout 25 days, about 2 days and about 30 days, about 3 days and about 30days, about 4 days and about 30 days, about 5 days and about 30 days,about 6 days and about 30 days, about 7 days and about 30 days, about 8days and about 30 days, about 9 days and about 30 days, about 10 daysand about 30 days, about 15 days and about 30 days, about 20 days andabout 30 days, or about 25 days and about 30 days. In anotherembodiment, a culture is maintained in production phase for at leastabout 1 day, at least about 2 days, at least about 3 days, at leastabout 4 days, at least about 5 days, at least about 6 days, at leastabout 7 days, at least about 8 days, at least about 9 days, at leastabout 10 days, at least about 11 days , at least about 12 days, at leastabout 15 days, at least about 20 days, at least about 25 days, or atleast about 30 days. In a further embodiment, a culture is maintained inproduction phase for about 1 day, about 2 days, about 3 days, about 4days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 11 days, about 12 days, about 15 days, about20 days, about 25 days, or about 30 days.

The present invention further provides a method of producing arecombinant glycoprotein interest, comprising culturing cells engineeredto express the recombinant glycoprotein of interest in a culturecomprising a medium described herein; and recovering or isolating therecombinant glycoprotein of interest from the culture. In certainembodiments, the recombinant glycoprotein of interest is an antibody ora fragment thereof. In a specific embodiment, the recombinantglycoprotein of interest is an anti-α4-integrin antibody. In anotherembodiment, the recombinant glycoprotein of interest is natalizumab.

In a specific embodiment, a method of producing a recombinantglycoprotein of interest according to the present invention produces amaximum glycoprotein titer of at least about 0.05 g/L , at least about0.1 g/L, at least about 0.25 g/L, at least about 0.5 g/L, at least about0.75 g/L, at least about 1.0 g/L, at least about 1.5 g/L, at least about2 g/liter, at least about 2.5 g/liter, at least about 3 g/liter, atleast about 3.5 g/liter, at least about 4 g/liter, at least about 4.5g/liter, at least about 5 g/liter, at least about 6 g/liter, at leastabout 7 g/liter, at least about 8 g/liter, at least about 9 g/liter, orat least about 10 g/liter. In another embodiment, the method accordingto the present invention produces a maximum glycoprotein titer ofbetween about 1 g/liter and about 10 g/liter, about 1.5 g/liter andabout 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter,about 4 g/liter and about 10 g/liter, about 5 g/liter and about 10g/liter, about 1 g/liter and about 5 g/liter, about 1 g/liter and about4.5 g/liter, or about 1 g/liter and about 4 g/liter. In a specificembodiment, the glycoprotein is an antibody. In another embodiment, theglycoprotein is a blood clotting factor.

The invention further provides a conditioned cell culture mediumproduced by a method described herein.

In one embodiment, a conditioned cell culture medium produced accordingto the provided methods comprises a recombinant glycoprotein ofinterest. In a specific embodiment, a conditioned cell culture mediumaccording to the invention comprises a recombinant glycoprotein ofinterest at a titer of at least about 2 g/liter, at least about 2.5g/liter, at least about 3 g/liter, at least about 3.5 g/liter, at leastabout 4 g/liter, at least about 4.5 g/liter, at least about 5 g/liter,at least about 6 g/liter, at least about 7 g/liter, at least about 8g/liter, at least about 9 g/liter, or at least about 10 g/liter, or atiter of between about 1 g/liter and about 10 g/liter, about 1.5 g/literand about 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter,about 4 g/liter and about 10 g/liter, about 5 g/liter and about 10g/liter, about 1 g/liter and about 5 g/liter, about 1 g/liter and about4.5 g/liter, or about 1 g/liter and about 4 g/liter. In anotherembodiment, a conditioned cell culture medium according to the inventioncomprises a recombinant glycoprotein at a higher titer than the titerobtained without the use of a medium described herein. In a specificembodiment, the protein or polypeptide is an antibody.

Anti-α4-Integrin Antibodies

Given the large number of antibodies currently in use or underinvestigation as pharmaceutical or other commercial agents, productionof antibodies is of particular interest in accordance with the presentinvention. Antibodies are proteins that have the ability to specificallybind a particular antigen. Any anti-α4-integrin antibody that can beexpressed in a host cell can be used in accordance with the presentinvention. In one embodiment, the anti-α4-integrin antibody to beexpressed is a monoclonal antibody.

Particular anti-α4-integrin antibodies can be made, for example, bypreparing and expressing synthetic genes that encode the recited aminoacid sequences or by mutating human germline genes to provide a genethat encodes the recited amino acid sequences. Moreover, theseantibodies can be produced, e.g., using one or more of the followingmethods.

Numerous methods are available for obtaining antibodies, particularlyhuman antibodies. One exemplary method includes screening proteinexpression libraries, e.g., phage or ribosome display libraries. Phagedisplay is described, for example, U.S. Pat. No. 5,223,409; Smith (1985)Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. Thedisplay of Fab's on phage is described, e.g., in U.S. Pat. Nos.5,658,727; 5,667,988; and 5,885,793.

In addition to the use of display libraries, other methods can be usedto obtain an antibody. For example, a protein or a peptide thereof canbe used as an antigen in a non-human animal, e.g., a rodent, i.e., amouse, hamster, or rat.

In one embodiment, the non-human animal includes at least a part of ahuman immunoglobulin gene. For example, it is possible to engineer mousestrains deficient in mouse antibody production with large fragments ofthe human Ig loci. Using the hybridoma technology, antigen-specificmonoclonal antibodies derived from the genes with the desiredspecificity can be produced and selected. See, e.g., XENOMOUSE™, Greenet al. (1994) Nature Genetics 7:13-21, U.S. 2003-0070185, WO 96/34096,and WO 96/33735.

In another embodiment, a monoclonal anti-α4-integrin antibody isobtained from the non-human animal, and then modified, e.g., humanizedor deimmunized. Winter describes an exemplary CDR-grafting method thatcan be used to prepare humanized antibodies described herein (U.S. Pat.No. 5,225,539). All or some of the CDRs of a particular human antibodycan be replaced with at least a portion of a non-human antibody. In oneembodiment, it is only necessary to replace the CDRs required forbinding or binding determinants of such CDRs to arrive at a usefulhumanized antibody that binds to an antigen.

Humanized anti-α4-integrin antibodies can be generated by replacingsequences of the Fv variable region that are not directly involved inantigen binding with equivalent sequences from human Fv variableregions. General methods for generating humanized antibodies areprovided by Morrison, S. L. (1985) Science 229:1202-1207, by Oi et al.(1986) BioTechniques 4:214, and by U.S. Pat. No. 5,585,089; U.S. Pat.No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 5,859,205; andU.S. Pat. No. 6,407,213. Those methods include isolating, manipulating,and expressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, can be obtained from a hybridoma producing anantibody against a predetermined target, as described above, fromgermline immunoglobulin genes, or from synthetic constructs. Therecombinant DNA encoding the humanized antibody can then be cloned intoan appropriate expression vector. In one embodiment, the expressionvector comprises a polynucleotide encoding a glutamine synthetasepolypeptide. (See, e.g., Porter et al., Biotechnol Prog 26(5):1446-54(2010).)

The anti-α4-integrin antibody can include a human Fc region, e.g., awild-type Fc region or an Fc region that includes one or morealterations. In one embodiment, the constant region is altered, e.g.,mutated, to modify the properties of the antibody (e.g., to increase ordecrease one or more of: Fc receptor binding, antibody glycosylation,the number of cysteine residues, effector cell function, or complementfunction). For example, the human IgG1 constant region can be mutated atone or more residues, e.g., one or more of residues 234 and 237.Antibodies can have mutations in the CH2 region of the heavy chain thatreduce or alter effector function, e.g., Fc receptor binding andcomplement activation. For example, antibodies can have mutations suchas those described in U.S. Pat. Nos. 5,624,821 and 5,648,260. Antibodiescan also have mutations that stabilize the disulfide bond between thetwo heavy chains of an immunoglobulin, such as mutations in the hingeregion of IgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol.Immunol. 30:105-08). See also, e.g., U.S. 2005-0037000.

In other embodiments, the anti-α4-integrin antibody can be modified tohave an altered glycosylation pattern (i.e., altered from the originalor native glycosylation pattern). As used in this context, “altered”means having one or more carbohydrate moieties deleted, and/or havingone or more glycosylation sites added to the original antibody. Additionof glycosylation sites to the presently disclosed antibodies can beaccomplished by altering the amino acid sequence to containglycosylation site consensus sequences; such techniques are well knownin the art. Another means of increasing the number of carbohydratemoieties on the antibodies is by chemical or enzymatic coupling ofglycosides to the amino acid residues of the antibody. These methods aredescribed in, e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit.Rev. Biochem. 22:259-306. Removal of any carbohydrate moieties presenton the antibodies can be accomplished chemically or enzymatically asdescribed in the art (Hakimuddin et al. (1987) Arch. Biochem. Biophys.259:52; Edge et al. (1981) Anal. Biochem. 118:131; and Thotakura et al.(1987) Meth. Enzymol. 138:350). See, e.g., U.S. Pat. No. 5,869,046 for amodification that increases in vivo half-life by providing a salvagereceptor binding epitope.

The anti-α4-integrin antibodies can be in the form of full lengthantibodies, or in the form of fragments of antibodies, e.g., Fab,F(ab′)₂, Fd, dAb, and scFv fragments. Additional forms include a proteinthat includes a single variable domain, e.g., a camel or camelizeddomain. See, e.g., U.S. 2005-0079574 and Davies et al. (1996) ProteinEng. 9(6):531-7.

In one embodiment, the anti-α4-integrin antibody is an antigen-bindingfragment of a full length antibody, e.g., a Fab, F(ab′)2, Fv or a singlechain Fv fragment. Typically, the anti-α4-integrin antibody is a fulllength antibody. The anti-α4-integrin antibody can be a monoclonalantibody or a mono-specific antibody.

In another embodiment, the anti-α4-integrin antibody can be a human,humanized, CDR-grafted, chimeric, mutated, affinity matured,deimmunized, synthetic or otherwise in vitro-generated antibody, andcombinations thereof.

The heavy and light chains of the anti-α4-integrin antibody can besubstantially full-length. The protein can include at least one, andpreferably two, complete heavy chains, and at least one, and preferablytwo, complete light chains or can include an antigen-binding fragment(e.g., a Fab, F(ab′)2, Fv or a single chain Fv fragment). In yet otherembodiments, the antibody has a heavy chain constant region chosen from,e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE;particularly, chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, moreparticularly, IgG1 (e.g., human IgG1). Typically, the heavy chainconstant region is human or a modified form of a human constant region.In another embodiment, the antibody has a light chain constant regionchosen from, e.g., kappa or lambda, particularly, kappa (e.g., humankappa).

Cells

Any eukaryotic cell or cell type susceptible to cell culture can beutilized in accordance with the present invention. For example, plantcells, yeast cells, animal cells, insect cells, avian cells or mammaliancells can be utilized in accordance with the present invention. In oneembodiment, the eukaryotic cells are capable of expressing a recombinantprotein or are capable of producing a recombinant or reassortant virus.

Non-limiting examples of mammalian cells that can be used in accordancewith the present invention include BALB/c mouse myeloma line (NSO/1,ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, TheNetherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells ±DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCLS 1); TRI cells (Mather et al., Annals N.Y. Acad.Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). In one embodiment, the present invention is used in theculturing of and expression of polypeptides from CHO cell lines. In aspecific embodiment, the CHO cell line is the DG44 CHO cell line. In aspecific embodiment, the CHO cell line is the DUXB11 CHO cell line. In aspecific embodiment, the CHO cell line comprises a vector comprising apolynucleotide encoding a glutamine synthetase polypeptide. In a furtherspecific embodiment, the CHO cell line expresses an exogenous glutaminesynthetase gene. (See, e.g., Porter et al., Biotechnol Prog26(5):1446-54 (2010).)

Additionally, any number of commercially and non-commercially availablehybridoma cell lines that express polypeptides or proteins can beutilized in accordance with the present invention. One skilled in theart will appreciate that hybridoma cell lines might have differentnutrition requirements and/or might require different culture conditionsfor optimal growth and polypeptide or protein expression, and will beable to modify conditions as needed.

The eukaryotic cells according to the present invention can be selectedor engineered to produce high levels of protein or polypeptide, or toproduce large quantities of virus. Often, cells are geneticallyengineered to produce high levels of protein, for example byintroduction of a gene encoding the recombinant glycoprotein of interestand/or by introduction of control elements that regulate expression ofthe gene (whether endogenous or introduced) encoding the recombinantglycoprotein of interest.

The eukaryotic cells can also be selected or engineered to survive inculture for extended periods of time. For example, the cells can begenetically engineered to express a polypeptide or polypeptides thatconfer extended survival on the cells. In one embodiment, the eukaryoticcells comprise a transgene encoding the Bc1-2 polypeptide or a variantthereof See, e.g., U.S. Pat. No. 7,785,880. In a specific embodiment,the cells comprise a polynucleotide encoding the bc1-xL polypeptide.See, e.g., Chiang G G, Sisk W P. 2005. Biotechnology and Bioengineering91(7):779-792.

The eukaryotic cells can also be selected or engineered to modify itsposttranslational modification pathways. In one embodiment, the cellsare selected or engineered to modify a protein glycolsylation pathway.In a specific embodiment, the cells are selected or engineered toexpress an aglycosylated protein, e.g., an aglycosylated recombinantantibody. In another specific embodiment, the cells are selected orengineered to express an afucosylated protein, e.g., an afucosylatedrecombinant antibody.

The eukaryotic cells can also be selected or engineered to allowculturing in serum free medium.

Media

The cell culture of the present invention is prepared in any mediumsuitable for the particular cell being cultured. In some embodiments,the medium contains e.g., inorganic salts, carbohydrates (e.g., sugarssuch as glucose, galactose, maltose or fructose), amino acids, vitamins(e.g., B group vitamins (e.g., B12), vitamin A vitamin E, riboflavin,thiamine and biotin), fatty acids and lipids (e.g., cholesterol andsteroids), proteins and peptides (e.g., albumin, transferrin,fibronectin and fetuin), serum (e.g., compositions comprising albumins,growth factors and growth inhibitors, such as, fetal bovine serum,newborn calf serum and horse serum), trace elements (e.g., zinc, copper,selenium and tricarboxylic acid intermediates), hydrolysates (hydrolyzedproteins derived from plant or animal sources), and combinationsthereof. Commercially available media such as 5×-concentrated DMEM/F12(Invitrogen), CD OptiCHO feed (Invitrogen), CD EfficientFeed(Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed (IrvineScientific), BD Recharge (Becton Dickinson), Cellvento Feed (EMDMillipore), Ex-cell CHOZN Feed (Sigma-Aldrich), CHO Feed BioreactorSupplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria),ActiCHO (PAA/GE Healthcare), Ham's F10 (Sigma), Minimal Essential Medium([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ([DMEM], Sigma) are exemplary nutrient solutions. In addition,any of the media described in Ham and Wallace,(1979) Meth. Enz., 58:44;Barnes and Sato,(1980) Anal. Biochem., 102:255; U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 5,122,469 or 4,560,655; InternationalPublication Nos. WO 90/03430; and WO 87/00195; the disclosures of all ofwhich are incorporated herein by reference, can be used as culturemedia. Any of these media can be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as gentamycin), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range) lipids (such as linoleic or other fatty acids) andtheir suitable carriers, and glucose or an equivalent energy source. Insome embodiments the nutrient media is serum-free media, a protein-freemedia, or a chemically defined media. Any other necessary supplementscan also be included at appropriate concentrations that would be knownto those skilled in the art.

In one embodiment, the mammalian host cell is a CHO cell and a suitablemedium contains a basal medium component such as a DMEM/HAM F-12 basedformulation (for composition of DMEM and HAM F12 media, see culturemedia formulations in American Type Culture Collection Catalogue of CellLines and Hybridomas, Sixth Edition, 1988, pages 346-349) with modifiedconcentrations of some components such as amino acids, salts, sugar, andvitamins, recombinant human insulin, hydrolyzed peptone, such asPrimatone HS or Primatone RL (Sheffield, England), or the equivalent; acell protective agent, such as Pluronic F68 or the equivalent pluronicpolyol; gentamycin; and trace elements. In another embodiment, thesuitable medium contains yeast hydrolysate. In a preferred embodiment,the suitable medium contains yeastolate.

The present invention provides a variety of media formulations that,when used in accordance with other culturing steps described herein,minimize or prevent decreases in cellular viability in the culture whileretaining the ability to control glycosylation of a recombinantglycoprotein of interest.

A media formulation of the present invention that has been shown to beto useful in manipulating glycosylation, while not having greatlynegative impacts on metabolic balance, cell growth and/or viability oron expression of polypeptide or protein comprises the media supplementdescribed herein. One of ordinary skill in the art will understand thatthe media formulations of the present invention encompass both definedand non-defined media.

Cell Culture Processes

Various methods of preparing mammalian cells for production of proteinsor polypeptides by batch and fed-batch culture are well known in theart. A nucleic acid sufficient to achieve expression (typically a vectorcontaining the gene encoding the polypeptide or protein of interest andany operably linked genetic control elements) can be introduced into thehost cell line by any number of well-known techniques. Typically, cellsare screened to determine which of the host cells have actually taken upthe vector and express the polypeptide or protein of interest.Traditional methods of detecting a particular polypeptide or protein ofinterest expressed by mammalian cells include but are not limited toimmunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, SDS-PAGE, Western blots, enzyme-linkedimmunosorbentassay (ELISA), high performance liquid chromatography(HPLC) techniques, biological activity assays and affinitychromatography. One of ordinary skill in the art will be aware of otherappropriate techniques for detecting expressed polypeptides or proteins.If multiple host cells express the polypeptide or protein of interest,some or all of the listed techniques can be used to determine which ofthe cells expresses that polypeptide or protein at the highest levels.

Once a cell that expresses the polypeptide or protein of interest hasbeen identified, the cell is propagated in culture by any of the varietyof methods well-known to one of ordinary skill in the art. The cellexpressing the polypeptide of interest is typically propagated bygrowing it at a temperature and in a medium that is conducive to thesurvival, growth and viability of the cell. The initial culture volumecan be of any size, but is often smaller than the culture volume of theproduction bioreactor used in the final production of the polypeptide orprotein of interest, and frequently cells are passaged several times inbioreactors of increasing volume prior to seeding the productionbioreactor. The cell culture can be agitated or shaken to increaseoxygenation of the medium and dispersion of nutrients to the cells.Alternatively or additionally, special sparging devices that are wellknown in the art can be used to increase and control oxygenation of theculture. In accordance with the present invention, one of ordinary skillin the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor, including butnot limited to pH, temperature, oxygenation, etc.

The cell density useful in the methods of the present invention can bechosen by one of ordinary skill in the art. In accordance with thepresent invention, the cell density can be as low as a single cell perculture volume. In some embodiments of the present invention, startingcell densities (seed density) can range from about 2×10² viable cellsper mL to about 2×10³, 2×10⁴, 2×10⁵, 2×10⁶, 5×10⁶ or 10×10⁶ viable cellsper mL and higher.

In accordance with the present invention, a cell culture size can be anyvolume that is appropriate for production of polypeptides. In oneembodiment, the volume of the cell culture is at least 500 liters. Inother embodiments, the volume of the production cell culture is 10, 50,100, 250, 1000, 2000, 2500, 5000, 8000, 10,000, 12,000 liters or more,or any volume in between. For example, a cell culture will be 10 to5,000 liters, 10 to 10,000 liters, 10 to 15,000 liters, 50 to 5,000liters, 50 to 10,000 liters, or 50 to 15,000 liters, 100 to 5,000liters, 100 to 10,000 liters, 100 to 15,000 liters, 500 to 5,000 liters,500 to 10,000 liters, 500 to 15,000 liters, 1,000 to 5,000 liters, 1,000to 10,000 liters, or 1,000 to 15,000 liters. Or a cell culture will bebetween about 500 liters and about 30,000 liters, about 500 liters andabout 20,000 liters, about 500 liters and about 10,000 liters, about 500liters and about 5,000 liters, about 1,000 liters and about 30,000liters, about 2,000 liters and about 30,000 liters, about 3,000 litersand about 30,000 liters, about 5,000 liters and about 30,000 liters, orabout 10,000 liters and about 30,000 liters, or a cell culture will beat least about 500 liters, at least about 1,000 liters, at least about2,000 liters, at least about 3,000 liters, at least about 5,000 liters,at least about 10,000 liters, at least about 15,000 liters, or at leastabout 20,000 liters.

One of ordinary skill in the art will be aware of and will be able tochoose a suitable culture size for use in practicing the presentinvention. The production bioreactor for the culture can be constructedof any material that is conducive to cell growth and viability that doesnot interfere with expression or stability of the produced polypeptideor protein.

The temperature of the cell culture will be selected based primarily onthe range of temperatures at which the cell culture remains viable. Forexample, during the initial growth phase, CHO cells grow well at 37° C.In general, most mammalian cells grow well within a range of about 25°C. to 42° C.

In certain cases, it can be beneficial or necessary to supplement thecell culture during the growth and/or subsequent production phase withnutrients or other medium components that have been depleted ormetabolized by the cells. For example, it might be advantageous tosupplement the cell culture with nutrients or other medium componentsobserved to have been depleted. Alternatively or additionally, it can bebeneficial or necessary to supplement the cell culture prior to thesubsequent production phase. As non-limiting examples, it can bebeneficial or necessary to supplement the cell culture with hormonesand/or other growth factors, particular ions (such as sodium, chloride,calcium, magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), amino acids, lipids, or glucose or otherenergy source.

These supplementary components, including the amino acids, can all beadded to the cell culture at one time, or they can be provided to thecell culture in a series of additions. In one embodiment of the presentinvention, the supplementary components are provided to the cell cultureat multiple times in proportional amounts. In another embodiment, it canbe desirable to provide only certain of the supplementary componentsinitially, and provide the remaining components at a later time. In yetanother embodiment of the present invention, the cell culture is fedcontinually with these supplementary components.

In accordance with the present invention, the total volume added to thecell culture should optimally be kept to a minimal amount. For example,the total volume of the medium or solution containing the supplementarycomponents added to the cell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume of the cell cultureprior to providing the supplementary components.

The cell culture can be agitated or shaken during the subsequentproduction phase in order to increase oxygenation and dispersion ofnutrients to the cells. In accordance with the present invention, one ofordinary skill in the art will understand that it can be beneficial tocontrol or regulate certain internal conditions of the bioreactor duringthe subsequent growth phase, including but not limited to pH,temperature, oxygenation, etc. For example, pH can be controlled bysupplying an appropriate amount of acid or base and oxygenation can becontrolled with sparging devices that are well known in the art.

In certain embodiments of the present invention, the practitioner canfind it beneficial or necessary to periodically monitor particularconditions of the growing cell culture. Monitoring cell cultureconditions allows the practitioner to determine whether the cell cultureis producing recombinant polypeptide or protein at suboptimal levels orwhether the culture is about to enter into a suboptimal productionphase.

In order to monitor certain cell culture conditions, it will benecessary to remove small aliquots of the culture for analysis. One ofordinary skill in the art will understand that such removal canpotentially introduce contamination into the cell culture, and will takeappropriate care to minimize the risk of such contamination.

As non-limiting example, it can be beneficial or necessary to monitortemperature, pH, cell density, cell viability, integrated viable celldensity, lactate levels, ammonium levels, osmolarity, or titer of theexpressed polypeptide or protein. Numerous techniques are well known inthe art that will allow one of ordinary skill in the art to measurethese conditions. For example, cell density can be measured using ahemacytometer, a Coulter counter, or Cell density examination (CEDEX).Viable cell density can be determined by staining a culture sample withTrypan blue. Since only dead cells take up the Trypan blue, viable celldensity can be determined by counting the total number of cells,dividing the number of cells that take up the dye by the total number ofcells, and taking the reciprocal. HPLC can be used to determine thelevels of lactate, ammonium or the expressed polypeptide or protein.Alternatively, the level of the expressed polypeptide or protein can bedetermined by standard molecular biology techniques such as coomassiestaining of SDS-PAGE gels, Western blotting, Bradford assays, Lowryassays, Biuret assays, and UV absorbance. It can also be beneficial ornecessary to monitor the post-translational modifications of theexpressed polypeptide or protein, including phosphorylation andglycosylation.

The practitioner can also monitor the metabolic status of the cellculture, for example, by monitoring the glucose, lactate, ammonium, andamino acid concentrations in the cell culture, as well as by monitoringthe oxygen production or carbon dioxide production of the cell culture.For example, cell culture conditions can be analyzed by using NOVABioprofile 100 or 400 (NOVA Biomedical, Wash.). Additionally, thepractitioner can monitor the metabolic state of the cell culture bymonitoring the activity of mitochondria. In one embodiment,mitochondrial activity can be monitored by monitoring the mitochondrialmembrane potential using Rhodamine 123. Johnson L V, Walsh M L, Chen LB. 1980. Proceedings of the National Academy of Sciences 77(2):990-994.

Isolation of Expressed Polypeptide

In general, it will typically be desirable to isolate and/or purifyproteins or polypeptides expressed according to the present invention.In one embodiment, the expressed polypeptide or protein is secreted intothe medium and thus cells and other solids can be removed, as bycentrifugation or filtering for example, as a first step in thepurification process.

Alternatively, the expressed polypeptide can be bound to the surface ofthe host cell. In this embodiment, the media is removed and the hostcells expressing the polypeptide or protein are lysed as a first step inthe purification process. Lysis of mammalian host cells can be achievedby any number of means well known to those of ordinary skill in the art,including physical disruption by glass beads and exposure to high pHconditions.

The polypeptide can be isolated and purified by standard methodsincluding, but not limited to, chromatography (e.g., ion exchange,affinity, size exclusion, and hydroxyapatite chromatography), gelfiltration, centrifugation, or differential solubility, ethanolprecipitation or by any other available technique for the purificationof proteins (See, e.g., Scopes, Protein Purification Principles andPractice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S. J.and Hames, B. D. (eds.), Protein Expression: A Practical Approach,Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J.N. (eds.), Guide to Protein Purification: Methods in Enzymology (Methodsin Enzymology Series, Vol 182), Academic Press, 1997, all incorporatedherein by reference). For immunoaffinity chromatography in particular,the protein can be isolated by binding it to an affinity columncomprising antibodies that were raised against that protein and wereaffixed to a stationary support. Alternatively, affinity tags such as aninfluenza coat sequence, poly-histidine, or glutathione-S-transferasecan be attached to the protein by standard recombinant techniques toallow for easy purification by passage over the appropriate affinitycolumn. Protease inhibitors such as phenyl methyl sulfonyl fluoride(PMSF), leupeptin, pepstatin or aprotinin can be added at any or allstages in order to reduce or eliminate degradation of the polypeptide orprotein during the purification process. Protease inhibitors areparticularly desired when cells must be lysed in order to isolate andpurify the expressed polypeptide or protein. One of ordinary skill inthe art will appreciate that the exact purification technique will varydepending on the character of the polypeptide or protein to be purified,the character of the cells from which the polypeptide or protein isexpressed, and the composition of the medium in which the cells weregrown.

Pharmaceutical Compositions

A polypeptide can be formulated as a pharmaceutical composition foradministration to a subject, e.g., to treat or prevent a disorder ordisease. Typically, a pharmaceutical composition includes apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Thecomposition can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (See e.g., Berge, S. M., etal. (1977) J. Pharm. Sci. 66:1-19). In one embodiment, a pharmaceuticalcomposition is an immunogenic composition comprising a virus produced inaccordance with methods described herein.

Pharmaceutical formulation is a well-established art, and is furtherdescribed, e.g., in Gennaro (ed.), Remington. The Science and Practiceof Pharmacy, 20^(th) ed., Lippincott, Williams & Wilkins (2000) (ISBN:0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7^(th) Ed., Lippincott Williams & Wilkins Publishers (1999)(ISBN: 0683305727); and Kibbe (ed.), Handbook of PharmaceuticalExcipients American Pharmaceutical Association, 3^(rd) ed. (2000) (ISBN:091733096X).

The pharmaceutical compositions can be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The form can depend on the intended mode of administration andtherapeutic application. Typically compositions for the agents describedherein are in the form of injectable or infusible solutions.

In one embodiment, the antibody is formulated with excipient materials,such as sodium chloride, sodium dibasic phosphate heptahydrate, sodiummonobasic phosphate, and a stabilizer. It can be provided, for example,in a buffered solution at a suitable concentration and can be stored at2-8° C.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze drying that yield a powder of an agent describedherein plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the polypeptide can be prepared with a carrierthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known. See, e.g., Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York(1978).

The foregoing description is to be understood as being representativeonly and is not intended to be limiting. Alternative methods andmaterials for implementing the invention and also additionalapplications will be apparent to one of skill in the art, and areintended to be included within the accompanying claims.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No:4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, Mass.(1984); and Steward, M. W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al. (eds), Basic and Clinical-Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W. H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunology4^(th) ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A.Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D.,Immunology 6th ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier HealthSciences Division (2005); Kontermann and Dubel, Antibody Engineering,Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII,Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR PrimerCold Spring Harbor Press (2003).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

EXAMPLES

During the natalizumab 2^(nd) Generation (“T2G”) manufacturing process,variability in drug substance pI isoforms and % Galactosylation (“Gal”)was observed. As shown in FIG. 1, % Gal values trended high and wereclose to the upper specification limit of 31.8% for several batches. Ananalysis of the 11 charge variant peaks of the antibody showed that Peak5 exhibited the most variation and was likely driving the shifts in therelative levels of the other charge variant peaks. Furthermore, % Galwas observed to trend inversely proportional with % Peak 5 (FIG. 1),making it a peak of interest for predicting Gal values.

Observed and predicted values for the pool peak isoforms of the antibodyin MFM-133-12-R060 were compared (FIG. 2). The modeling showed thatvariation in Peak 5 content was very well described by cell culturein-process data, particularly by lactate and pH values from theproduction bioreactor.

In the T2G manufacturing process, the production bioreactor pH wascontrolled at 7.0±0.2 through the addition of CO₂ (upper deadband) andNa₂CO₃ (lower deadband). Culture pH, which is directly impacted by cellnet lactate production, drifted between the upper and lower controllimits through the production run. As demonstrated through modeling,large-scale manufacturing (LSM) trends showed that an increase in Peak5/decrease in Gal levels could be correlated to a decrease in celllactate production/increase in Day 8 culture pH (FIGS. 3A-3D).

Based on this understanding, small-scale studies were carried out toidentify implementable methods for controlling product quality,specifically Peak 5 and Gal levels. The methods tested aimed to modulatelactate and pH levels in the production culture. Studies were alsocarried out concurrently to identify sources of the observed productquality variability, particularly relating to TC yeastolate lot-to-lotvariation. It was found that copper addition is an effective approachfor controlling Peak 5 and Gal levels. Further, it was demonstrated thatcopper variation in yeastolate is a significant source of productquality variability through analytical testing and supporting laboratoryresults.

Example 1 Effect of Copper (II) Sulfate on Cell Culture

The impact of different copper levels in the culture on cell metabolismand Peak 5/Gal was evaluated in shake flask studies. In four separateexperiments, varying amounts of copper (II) sulfate was added to theproduction culture on Day 0 and cells were cultured under different CO₂conditions. FIG. 4A shows a plot of %Peak 5 against Day 8 pH with copperaddition. FIG. 4B shows a plot of %Gal against Day 8 pH with copperaddition. Day 8 pH was chosen as a time point primarily because it is agood marker of the lactate metabolism of the culture.

Cell net lactate production decreased as copper was added to theculture. Since culture pH is directly driven by lactate production, anincrease in pH was observed. Increase in pH levels due to copperaddition correlated with an increase in % Peak 5 (R²=0.95) and decreasein % Gal (R²=0.91). The correlation found in the small-scale experimentsalso aligned well with data gathered from large-scale manufacturing asshown in FIGS. 4A and 4B.

Example 2 Trace Copper Concentrations in Yeastolate

An analysis of LSM data showed that within a single lot of TC yeastolateused for two batches (108864), consistent viable cell density, pH andlactate concentration profiles were observed (TR-MS-000030). Using X-RayFluorescence (XRF) and ICP-MS, trace metal concentrations present in 20yeastolate lots were also measured.

In XRF, X-ray photons of sufficient energy strike an atom and dislodgean electron from one of the inner electron orbitals, typically the Kand/or L shell. To regain stability, an electron from one of the outerorbitals fills this vacancy and, in the process, excess energy isreleased in the form of an X-ray photon. Because the quantum states ofeach atom's electrons are fairly unique, the energy of the emittedphotons are characteristic of the elements present and the number ofphotons detected at a specific energy is proportional to theconcentration of that element in the sample. Palmer, P. T. et al., J.Agr. Food Chem. 57(7):2605-2613 (2009).

ICP-MS (inductively coupled plasma mass spectrometry) is a type of massspectrometry. ICP-MS mostly utilizes noble gases, such as argon asplasma gas, in which the efficient vaporization, dissociation oratomization, excitation, and final ionization of the sample constituentsto be analyzed takes place. In addition, this high temperature processleads to a complete fragmentation of every sample molecule, leaving onlytheir detectable, atomic constituents, which could then be used assurrogates to also detect complex molecules. Pröfrock, D. and Prange,A., Appl. Spectr. 66(8):843-868 (2012).

Copper concentrations were found to vary from approximately 0.6 to 2.3ppm among the different yeastolate lots. Strong correlations (R²>0.58)were observed between the measured copper levels in yeastolate to LSMcell culture lactate/pH levels and Peak 5/Gal levels, as shown in FIGS.5A-5D.

Example 3 Other Sources of Trace Copper Contamination

Trace copper contamination was measured and detected in most of theother raw materials used in the T2G manufacturing process, but at verylow levels. An initial screen on a wide set of raw materials (NMB1,IMF2.0v3, Sodium Bicarbonate, L-Tyrosine, Tropolone, Ammonium FerricCitrate Brown, HEPES, D-Glucose, Gibco Cholesterol Lipid Concentrate)was performed by XRF. Raw materials (IMF and Sodium Bicarbonate) whichshowed significant copper levels were submitted for ICP-MS analysis toquantify concentrations. Table 1 highlights the copper levels in twolots of IMF2.0v3 and NaHCO₃ powder, and their correspondingconcentrations in the media, feed, and culture; the two lots shown foreach raw material each had the highest and lowest copper concentrationsout of 6 to 8 lots measured. Copper concentrations from yeastolate werealso listed for comparison. It should be noted that the IMF formulationhad a target copper concentration of 80 nM, which accounts for 73.3 nMof the copper measured in the prepared nutrient feed.

TABLE 1 Copper Concentration in powder, Calculated Copper Concentrationmeasured by Nutrient Day 0 Day 9 Raw ICP-MS Media Feed Culture CultureMaterial (ppm) (nM) (nM) (nM) (nM) Yeastolate R08543 0.675 13.3 607.413.3 98.3 R08216 2.251 44.3 2025.5 44.3 327.9 IMF2.0v3 R09573 0.09 —134.9 0 18.9 R08496 0.14 — 209.9 0 29.4 NaHCO₃ R08658 0.02 0.73 — 0.730.73 C41485 0.05 1.81 — 1.81 1.81

The reported data provided a magnitude of the contribution of each rawmaterial to additional copper in the culture, and of the coppervariation among different lots. It was observed that trace coppercontamination originating from IMF2.0v3 and NaHCO₃ were very low whencompared to that from yeastolate.

Media and nutrient feed retains were also sent for external ICP-MStesting to evaluate other potential sources of copper contamination andconsistency in the copper levels between Facilities A and B. Copperlevels were below the limit of detection in the media samples. FIG. 6shows the copper concentration measured in the nutrient feed for 3Facility A and Facility B batches using the same yeastolate lot. Themeasured copper levels were compared to the calculated levels based onmeasurements in the yeastolate powder.

No considerable differences were observed between sites or between themeasured and calculated copper concentrations in the nutrient feedsamples, demonstrating that yeastolate powder was the main source ofcopper contamination.

Example 4 Copper Supplementation to Yeastolate

Based on the XRF copper concentration measurements, the effects ofcopper supplementation to low-copper yesatolate lots on cell metabolismand Peak 5/Gal levels were evaluated in 1 L production shake flaskcultures. Yeastolate lots with 0.675 ppm (R08543) and 2.251 ppm (R08216)of measured copper concentrations were chosen. TC yeastolate R08543 wassupplemented with 1.576 ppm of additional copper (II) sulfate to matchthe amount of copper present in TC yeastolate R08216, and the impact ofcopper supplementation on cell growth, lactate, and pH are shown inFIGS. 7A-7C. The T2G culture media is made with 1.25 g/kg of yeastolatewhile the IMF nutrient, which is fed to cultures from days 3 to 11 at 2%volume, is made with 57.14 g/kg of yeastolate.

Significant differences were observed in cell metabolism and growthprofiles with cultures receiving yeastolates from the two differentlots. Copper supplementation to TC yeastolate R08543 signficantlyreduced lactate production, increased culture pH, and recovered the cellgrowth performance of the cultures in a manner similar to thosereceiving TC yeastolate R08216. With copper supplementation, Peak 5levels increased from an average of 8.2 to 13.4% and Gal levelsdecreased from an average of 25 to 15%, to match those of Lot R08216cultures (FIGS. 8A and 8B).

The same experiment was also carried out with yeast extracts from BioSpringer. Becton Dickinson (BD), which is the primary supplier of the TCyeastolate used in the T2G process. BD acquires yeast extract from BioSpringer and adds proprietary supplements to the yeast extracts beforesupplying them for use. Using XRF, copper concentrations in two lots ofyeast extracts were measured to be 0.519 ppm (Lot 1) and 1.084 ppm Lot2. Lot 1 was supplemented with 0.57 ppm of additional copper (II)sulfate to match the amount of copper present in Lot 2 (Lot 1+2× Cu).

As shown in FIGS. 9A-9C, copper supplementation reduced lactate levelsand caused an increase in both pH and growth in Lot 1+2× Cu cultures tomatch the profiles of Lot 2 very closely.

Similarly, an increase in Peak 5 levels from an average of 8.1 to 9.1%and a decrease in Gal levels from an average of 25 to 19.1% was observedwhen copper was added to Lot 1 (FIGS. 10A and 10B). The different copperlevels measured in Bio Springer yeast extracts also indicate that thevariation of this metal concentration is inherent to the non-chemicallydefined nature of the raw material.

Example 5 Methods for Controlling Cell Culture

Evidence through both large-scale data and small-scale studies suggestedthat the variation in copper levels among yeastolate lots is asignificant source of the observed product quality variability, and thatcontrolling copper levels within yeastolate would be an effective methodof modulating product quality.

A stress test was carried out with copper addition to yeastolate at highconcentrations to identify risks and failure points. In two separateexperiments, copper (II) sulfate was added at different concentrationsup to 6× and 10× copper in TC yeastolate R08543 (Control), respectively:

TABLE 2 Experiment 1 Experiment 2 Cu in TC yeastolate Cu in TCyeastolate Group (ppm) Group (ppm) Control 0.68 Control 0.68 2.2X Cu1.46 3.3X Cu   2.25 3.3X Cu 2.25 6X Cu 4.05   5X Cu 3.38 8X Cu 5.40   6XCu 4.05 10X Cu  6.75

In Experiment 1, no negative impact on cell growth and titer wereobserved up to 4.05 ppm Cu in yeastolate (FIG. 11A). In Experiment 2,the highest cell growth was observed at 3.3× Cu, with peak viable celldensity (VCD) reaching approximately 1.2E+07 vc/mL. A decline in thecell growth profile was observed as copper concentrations increased to8× and 10× Cu; nevertheless, the profiles were still comparable to thatof the control group (FIG. 11B). There was no significant differences intiter among all groups. The concentration of copper in yeastolatemeasured was no higher than 2.25 ppm while the experimentally testedcopper concentration was up to 6.75 ppm. This study demonstrated that nonegative growth or titer impact of copper would be expected when copperwas added to the yeastolate at the studied levels.

FIG. 12 shows the impact of high copper concentrations on Peak 5 andPeak 4. At large-scale, harvest material was processed over the TMAEcolumn which reduces Peak 5 (average 2.9%) while enriching Peak 4(average 6.9%). As laboratory samples were purified through a singleProtein A column, results were adjusted to account for Peak 5reduction/Peak 4 enrichment for a better comparison to LSM data.

While there is no specification limit for Peak 5, both LSM andexperimental data showed that increases in this peak content also drovea decrease in % Peak 4 towards its lower specification limit of 58.4%.Projection of this data set showed that at approximately 4 ppm Cu, Peak4 hits its lower limit, thereby placing a limit on Peak 5 at 15%.Increases in Peak 5 content at high copper concentrations also reduces %Gal, which has a lower specification limit of 13.4% (FIG. 13).

Experimental data showed that for the copper concentrations tested, Gallevels were never driven below the specification; minimal changes in Gallevels were observed above 3.5 ppm Cu. At 4 ppm Cu and the projectedPeak 5 limit of 15%, Gal decreased to approximately 15%.

Example 6 Copper Operating Range for Cell Culture

The highlighted region in FIG. 14 was proposed as the target copperoperating range. Stress tests indicated that a negative impact on growthor failure due to Peak 5/Peak 4 may occur above 4 ppm Cu in yeastolate.The proposed range remains with the range of variation and issignificantly below the failure point.

To arrive at this operating range, it was proposed that TC yeastolatelots be supplemented with copper when needed. An acceptable range ofcopper concentration in yeastolate would be provided. It would berequired that the copper concentration of each incoming lot of yeastextract would be measured through ICP-MS or XRF. All TC yeastolate lotswith copper levels within the range provided would be accepted. For lotswith copper levels below the acceptable range, it would be required thatthose lots be supplemented with copper (II) sulfate up to a targetconcentration, the lots be retested, and the final concentrationsreported. Lots with copper levels above the controlled range would notbe used. This method would allow for a tighter control of the rawmaterial over an operating range that remains within experience.

Example 7 Additional Experiments Studying the Impact of Copper on T2GProduct Quality

Experiments were carried out in 1 L shake flasks and levels of copper inyeastolate were manipulated to be between 0.6 ppm and 6.8 ppm. All LSMvalues were drug substance data while laboratory samples were purifiedthrough a single Protein A column. FIGS. 15A and 15B show no impact ofincreasing copper levels on % aggregate and % half antibody.

FIGS. 16A-16J shows plots of acidic and basic pI isoforms against copperconcentration. Copper had no impact on acidic isoforms (Peaks 1 to 3).With the exception of Peaks 7 and 11, copper was observed to impact allother basic isoforms.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All documents, articles, publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

What is claimed is:
 1. A method for achieving a predeterminedgalactosylation profile of an anti-α4-integrin antibody comprisingadjusting the concentration of copper in a cell culture to achieve atarget copper concentration range, wherein the cell culture compriseshost cells producing the anti-α4-integrin antibody.
 2. The method ofclaim 1, comprising supplementing the cell culture with copper if thecopper concentration in the cell culture is below the target copperconcentration range.
 3. The method of claim 1 or 2, wherein thepredetermined galactosylation profile of the anti-α4-integrin antibodiescomprises 13 to 32% galactosylation.
 4. The method of any one of claims1-3, wherein the target copper concentration range in the cell cultureis 1.0 ppm to 2.4 ppm.
 5. The method of claim 4, wherein the targetcopper concentration range in the cell culture is at day 0 between 20 nmand 50 nM and at day 11 between 200 nM and 500 nM.
 6. The method of anyone of claims 1-5, wherein the target copper concentration is maintainedthrough a feedback loop.
 7. The method of claim 6, wherein the copperconcentration is constantly monitored and maintained within the targetcopper concentration range.
 8. A method for achieving a predeterminedgalactosylation profile of an anti-α4-integrin antibody comprising (i)determining a copper concentration in a component of a cell culturemedium, (ii) if the copper concentration is below a target copperconcentration range, supplementing the component of the cell culturemedium with copper to achieve a copper concentration within the targetcopper concentration range, (iii) producing a cell culture medium usingthe component of cell culture medium with the target copperconcentration, and (iv) culturing a recombinant host cell producing ananti-α4-integrin antibody in the cell culture medium comprising the cellculture medium component.
 9. A method for achieving a predeterminedgalactosylation profile of an anti-α4-integrin antibody comprising (i)determining a copper concentration in a component of a cell culturemedium, (ii) if the copper concentration is below a target copperconcentration range, adding copper to the component of the cell culturemedium to achieve a copper concentration within the target copperconcentration range, (iii) producing a cell culture medium using thecomponent of cell culture medium with the target copper concentration,and (iv) culturing a recombinant host cell producing an anti-α4-integrinantibody in the cell culture medium comprising the cell culture mediumcomponent with the target copper concentration.
 10. The method of claim8 or 9, wherein the predetermined galactosylation profile of theanti-α4-integrin antibodies comprises 13 to 32% galactosylation
 11. Themethod of claim 9 or 10, wherein the component of the cell culturemedium is a yeast hydrolysate.
 12. The method of claim 11, wherein thehydrolysate is yeastolate.
 13. The method of any one of claims 9-12,wherein the target copper concentration range in the component of thecell culture medium is 1.0 ppm to 2.4 ppm.
 14. The method of claim 13,wherein the target copper concentration range at day 0 between 20 nm and50 nM and at day 11 between 200 nM and 500 nM.
 15. The method of any oneof claim 1-8, or 10-11, wherein the target copper concentration isachieved with a single dose of copper.
 16. The method of any one ofclaims 1-15, wherein the anti-α4-integrin antibody is produced by aeukaryotic host cell.
 17. The method of claim 16, wherein the eukaryotichost cell is a mammalian host cell.
 18. The method of any one of claims1-17, wherein the anti-α4-integrin antibody is produced at amanufacturing scale.
 19. A method for optimizing a cell culture mediumfor the production of an anti-α4-integrin antibody comprising (i)determining the amount of copper in a cell culture medium or a componentused to produce a cell culture medium, and (ii) if the amount of copperis below a target range, supplementing the cell culture medium or thecomponent of the cell culture medium with copper to achieve an amount ofcopper within the target range, wherein the target range is sufficientto produce anti-α4-integrin antibodies with a predeterminedgalactosylation profile.
 20. The method of claim 19, wherein thepredetermined galactosylation profile of the anti-α4-integrin antibodiescomprises 13 to 32% galactosylation.
 21. The method of any one of claim3, 10, or 20, wherein the predetermined galactosylation profile of theanti-α4-integrin antibody comprises 13.4 to 31.8% galactosylation. 22.The method of claim 21, wherein the predetermined galactosylationprofile of the anti-α4-integrin antibody comprises 15 to 31%galactosylation.
 23. The method of claim 22, wherein the predeterminedgalactosylation profile of the anti-α4-integrin antibody comprises 25%galactosylation.
 24. The method of any one of claims 1-23, wherein thecopper concentration alters the levels of the isoform variants of theanti-α4-integrin antibody.
 25. A method for obtaining a component for acell culture medium comprising (i) measuring the amount of copper in ahydrolysate from yeast and (ii) if the amount of copper is below atarget range, supplementing the yeast hydrolysate with copper to achievean amount of copper within the target range.
 26. The method of claim 25,wherein the hydrolysate from yeast is yeastolate.
 27. The method ofclaim 26, wherein anti-α4-integrin antibodies produced by recombinanthost cells cultured in cell culture medium comprising the yeast lysatecomprise a predetermined galactosylation profile.
 28. The method ofclaim 27, wherein the predetermined galactosylation profile of theanti-α4-integrin antibody comprises 13 to 32% galactosylation.
 29. Themethod of any one of claims 1-24 and 27-28, wherein the anti-α4-integrinantibody is natalizumab.
 30. The method of any one of claims 1-29,wherein the copper used to supplement the cell culture, cell culturemedium component, or hydrolysate from yeast is copper (II) sulfate.